The role of cis-elements in the evolution of crassulacean acid metabolism photosynthesis

Abstract

Crassulacean acid metabolism (CAM) photosynthesis is an innovation of carbon concentrating mechanism that is characterized by nocturnal CO₂ fixation. Recent progresses in genomics, transcriptomics, proteomics, and metabolomics of CAM species yielded new knowledge and abundant genomic resources. In this review, we will discuss the pattern of cis-elements in stomata movement-related genes and CAM CO₂ fixation genes, and analyze the expression dynamic of CAM related genes in green leaf tissues. We propose that CAM photosynthesis evolved through the re-organization of existing enzymes and associated membrane transporters in central metabolism and stomatal movement-related genes, at least in part by selection of existing circadian clock cis-regulatory elements in their promoter regions. Better understanding of CAM evolution will help us to design crops that can thrive in arid or semi-arid regions, which are likely to expand due to global climate change.

Introduction

Photosynthesis is the process that harvests solar energy to synthesize organic compounds that can ultimately be utilized to drive cellular processes by all forms of life. Photosynthesis is known from cyanobacteria to their descendants including algae and vascular plants¹. There are three different photosynthetic pathways in terrestrial plants for fixation of carbon dioxide (CO₂): C₃, C₄, and CAM. C₃ photosynthesis is employed by most vascular plants. C₄ plants represent about 3% of vascular plants², while CAM plants represent about 6%³. Both C₄ and CAM are add-ons to the C₃ pathway. C₄ and CAM metabolisms are similar in biochemistry but CO₂ concentration steps are spatially separated in C₄ rather than temporally as in CAM. C₄ minimizes photorespiration by concentrating CO₂ in bundle sheath cells, which relies in part on the unique cellular structure (Fig. 1a). Many C₄ plants are agronomically important species, such as maize and sugarcane⁴. CAM plants have high water-use efficiency (WUE, expressed as mmol CO₂ mol⁻¹ H₂O), which is a direct consequence of the fact that they open their stomata at night and keep them closed during the daytime⁵. WUE for carbon assimilation in CAM plants is much higher than in C₃ or C₄ plants. It will be 2–10 times higher than that of C₄ plants and 2.6–20 times higher than that of C₃ plants⁶. One of the major differences between C₄ and CAM photosynthesis centers on the temporal regulation of CO₂ absorption and fixation (Fig. 1).

Fig. 1. Photosynthetic reactions in C₄ and CAM plants.

a NADP-malic enzyme type of C₄ pathway. b Carbon fixation in CAM plants.

CAM is found in over 400 genera across 36 families of vascular plants⁷ and has evolved multiple times independently from diverse ancestral C₃ plants³. While we know ecological factors such as drought condition and CO₂ concentration drive the evolution of CAM^8–10, far less is known about genetics. Gene family duplication was previously proposed as the driver of CAM metabolism evolution through neofunctionalization of newly duplicated paralogous genes³, while others proposed that C₄ and CAM photosynthesis may have arisen through the re-organization of metabolic processes already present in C₃ plants^11–13.

The past few years have seen the rapid progresses in genomics, transcriptomics, proteomics, and metabolomics for an increasing number of plant species including CAM species. The orchid Phalaenopsis equestris was the first CAM species for which the genome was assembled in 2015¹⁴. In the same year, the genome of fruit pineapple (Ananas comosus var. comosus) cultivar ‘F153’, which has been cultivated by Del Monte for 50 years, was sequenced, and the evolution of CAM photosynthesis was investigated¹⁵. In a following work, temporal and spatial transcriptomic profiles of CAM-performing mature leaves have also been studied in fruit pineapple¹⁶. Recently, we sequenced the bracteatus pineapple (Ananas comosus var. bracteatus) accession CB5 genome, and assembled to chromosomal level¹⁷. The genome of Kalanchoë fedtschenkoi, a eudicot CAM species was also available in 2017¹⁸. Furthermore, multi-dimensional omics data were available for CAM species, such as Agave americana^19,20, ochids^21,22, and Talinum triangulare²³. These progresses for species that evolved CAM independently provide an excellent resource for comparative analyses, which will help us have a better understanding on the evolution of CAM photosynthesis.

Cis-elements of stomatal movement-related genes

Stomata were first described as ‘pore-like’ structures on the surface of leaves over three centuries ago²⁴. Since the earliest examples of stomata were discovered in the leaf fossil record, plants have been evolving in terms of size and density of stomata to maintain the maximum leaf conductance as the atmosphere CO₂ changed²⁵. Stomata play an essential role in controlling of transpiration rate and water homeostasis in plants²⁶. Stomatal movement can be stimulated by different environmental factors, such as light, abscisic acid (ABA), pathogens, CO₂ and air humidity²⁷. Among them, air humidity and ABA are directly related to water status in plants²⁸. In CAM plants, the diel rhythms of stomatal conductance and transpiration are closely linked to the net CO₂-uptake rhythm⁵.

CAM plants present a reverse stomatal conductance pattern by assimilating CO₂ during the night when the temperature is low resulting in lower evapotranspiration rate compared to C₃ and C₄ plants^29,30. This unique pattern of stomatal movement leads to the higher WUE in CAM plants³¹. The reverse stomatal rhythm has aroused curiosity and investigation for centuries³². Understanding the regulation of stomatal movement-related genes in CAM species may provide promising opportunities for engineering crops with higher WUE³².

We identified 118 stomatal movement-related genes in A. comosus var. comosus, 95 in A. comosus var. bracteatus, 121 in P. equestris, 140 in Arabidopsis, 123 in rice, and 121 in sorghum (Supplementary Table S1). Based on the GO annotation, the stomatal movement-related genes were divided into three categories, including genes involved in stomatal opening, stomatal closure, and regulation of stomatal movement. For genes involved in stomatal movement, the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1)-binding site (CBS; AAAAATCT) and G-box binding site (CACGTG) showed more than 10% or higher frequency than the expected frequencies based on random chance in A. comosus var. comosus (Table 1). The G-box element was enriched in genes involved in all three categories in A. comosus var. comosus (Table 1). The evening element (EE; AAAATATC) and CBS were enriched in 123 stomatal movement-related genes in rice, whereas the morning element (MOE; CCACAC) was only enriched in stomatal opening category in rice (Table 1). When comparing with non-CAM species, Motif ERF73, ERF7, and ABR1 were enriched in CAM species (Supplementary Table S2). Based on these in silico findings, we propose the hypothesis that these different sets of cis elements regulate stomatal opening during the day and closure during the night for these three C₃ and C₄ species. Interestingly, stomatal movement-related genes in A. comosus var. comosus have higher frequencies of circadian clock cis-regulatory elements than A. comosus var. bracteatus (Table 1).

Table 1.

Frequency of circadian clock-associated motifs (per kb) in 2 kb promoter regions of genes involved in stomatal movement^a.

Circadian Motif	A. comosus var. comosus	A. comosus var. bracteatus	P. equestris	Arabidopsis	rice	sorghum
Genes involved in stomatal movementb
MOE	0.212 (0.250)	0.170 (0.269)	0.132 (0.152)	0.164 (0.215)	0.333 (0.367)	0.281 (0.327)
EE	0.110 (0.105)	0.101 (0.096)	0.107 (0.168)	0.125 (0.124)	0.106 (0.059)	0.074 (0.072)
CBS	0.148 (0.105)	0.160 (0.096)	0.087 (0.168)	0.118 (0.124)	0.069 (0.059)	0.058 (0.072)
G-box	0.288 (0.250)	0.250 (0.269)	0.120 (0.152)	0.232 (0.215)	0.317 (0.367)	0.248 (0.327)
Regulation of stomatal movement
MOE	0.209	0.180	0.156	0.174	0.276	0.256
EE	0.115	0.090	0.065	0.107	0.141	0.103
CBS	0.203	0.172	0.097	0.112	0.090	0.064
G-box	0.284	0.270	0.130	0.247	0.359	0.231
Stomatal closure
MOE	0.185	0.188	0.089	0.121	0.397	0.296
EE	0.093	0.094	0.214	0.136	0.069	0.037
CBS	0.074	0.188	0.071	0.152	0.034	0.074
G-box	0.296	0.000	0.089	0.152	0.259	0.352
Stomatal opening
MOE	0.250	0.206	0.088	0.184	0.471	0.353
EE	0.111	0.176	0.118	0.184	0.000	0.000
CBS	0.028	0.088	0.059	0.079	0.029	0.000
G-box	0.278	0.235	0.118	0.289	0.206	0.147

^aClock-associated motifs with ≥10% frequency higher than expected frequency appearance in stomatal movement-related genes than expected genome wide frequency is highlighted in bold

^bSpecies-specific expected frequencies are indicated in brackets. For example, in A. comosus var. comosus, since the genome GC content is 38%, G/C and A/T occurrence probabilities are 0.19 and 0.31. Both forward and reversed strands were included to calculate the expected frequency of the motif occurrence per kb (For MOE (CCACAC): 0.19 × 0.19 × 0.31 × 0.19 × 0.31 × 0.19 × 2 × 1,000 = 0.250)

In A. americana, the temporal re-programming of particular genes, including CO₂ and ABA signaling and turgor pressure regulating genes are essential to regulate stomatal movement¹⁹. Comparative transcriptomic analyses between the C₃ and CAM Erycina species also showed that genes involved in light and ABA signaling are altered²². The numbers of genes that contain cis-elements involved in several key stomatal movement pathways, such as light, ABA, and stress, are summarized (Table 2). ABA responsiveness-related motif (ABRE) appeared most frequently compared to other signaling pathways in the three species (Arabidopsis, P. equestris, and sorghum) with different photosynthetic pathways. From previous studies, exogenous ABA can induce stomatal closure and the expression and activity of CAM^33,34. Moreover, stress-related motif (STRE) was the most frequent in the stomatal related genes of rice and pineapple (Table 2). The stress-induced stomatal movement signaling pathway is closely related to the water status of the plant³⁵. Further genomic and molecular analysis of potential stomatal movement genes will enable us to have a comprehensive understanding of stomatal biology of CAM plants, and might provide candidate genes for engineering crop plants with higher sustainable production^32,36.

Table 2.

The number of genes and their percentages to the total genes of the genomes that contain cis-elements involved in partial key stomatal movement pathways annotated at promoter regions of orthologs in A. comosus var. comosus, A. comosus var. bracteatus, P. equestris, Arabidopsis, rice, and sorghum.

Pathways		A. comosus var. comosus	A. comosus var. bracteatus	P. equestris	Arabidopsis	rice	sorghum
Light responsiveness	Box 4a	107 (0.40%)	69 (0.23%)	30 (0.10%)	116 (0.24%)	89 (0.17%)	76 (0.22%)
	GT1-motifb	73 (0.27%)	42 (0.14%)	38 (0.13%)	101 (0.21%)	80 (0.15%)	61 (0.18%)
	GA-motifc	25 (0.09%)	11 (0.04%)	19 (0.06%)	29 (0.06%)	17 (0.03%)	33 (0.10%)
	G-Boxd	40 (0.15%)	18 (0.06%)	36 (0.12%)	40 (0.08%)	50 (0.10%)	105 (0.31%)
	TCT-motife	75 (0.28%)	46 (0.16%)	48 (0.16%)	112 (0.23%)	55 (0.10%)	56 (0.16)
ABA responsiveness	ABREf	101 (0.37%)	65 (0.22%)	58 (0.20%)	123 (0.25%)	115 (0.22%)	112 (0.33%)
Stress	WUN-motifg	29 (0.11%)	27 (0.09%)	42 (0.14%)	62 (0.13%)	47 (0.09%)	43 (0.13%)
	STREh	116 (0.43%)	89 (0 .30%)	35 (0.12%)	82 (0.17%)	116 (0.22%)	104 (0.30%)

^a Box 4-motif (ATTAAT)

^bGT1-motif (GGTTAA)

^cGA-motif (ATAGATAA)

^dG-Box (CACGTG)

^eTCT-motif (TCTTAC)

^fABRE-motif (ACGTG)

^gWUN-motif (AAATTACT)

^hSTRE motif (AGGGG)

Diurnal transcript abundance patterns of CAM pathway genes: pineapple as an example

The pineapple genome assembly also allowed the identification of full- and partial-length predicted amino-acid sequences of the key metabolic enzymes comprising the core carboxylation module of CAM responsible for nocturnal fixation of CO₂^15,31,37. Carbonic anhydrase (CA), catalyzing the conversion of CO₂ into HCO₃^–, is responsible for the first step in CO₂ assimilation both in C₄ and CAM plants. All three CA subfamily (α, β, and γ) enzymes were identified in pineapple genome (Supplementary Table S3). Only βCA genes (AccβCA2–1 and AccβCA2–2) implicated in CAM-specific roles due to their mRNA abundance in green leaf tissue¹⁵, indicating that βCA may acts as the enzyme in the initiation of CO₂ fixation.

Three genes encoding the key enzyme PPC responsible for nocturnal CO₂ fixation were identified in the genome assembly, all of which are predicted to be localized to the cytosol as expected¹⁵. Three PPC genes were identified in comosus pineapple genome (Supplementary Table S3, Supplementary Fig. S1). Among these three PPC genes, AccPPC1 is the most abundant transcript (>3000 FPKM, fragments per kilobase of exon per million fragments mapped) and displayed highest abundance at 6 pm (>5500 FPKM) in CAM-performing leaf tissues. In T. triangulare, a facultative CAM species, PPC was upregulated 25-fold (to 15,510 rpm, reads per million) at midnight on day 9 and 12 of water limitation when indicative of CAM was observed²³. Comparative transcriptomic analyses between the C₃ and CAM Erycina species also demonstrated that PPC gene in CAM Erycina displayed higher abundance than in C3 Erycina²². These results suggest that high levels of PPC transcripts are important for CAM.

PPC undergoes reversible N-terminal phosphorylation by a circadian clock-controlled PPC kinase (PPCK), which reduces the sensitivity of the enzyme to allosteric inhibition by L-malate and increases its affinity for its substrate phosphoenolpyruvate (PEP)^38,39. In A. americana, which is an obligate CAM plant, PPCK1 gene displayed diel transcripts abundance pattern, suggesting its important role in temporal re-programming of CAM²⁰. In K. fedtschenkoi, PPCK1 is also essential for nocturnal CO₂ fixation; moreover, knock-down of oscillations in the transcript abundance of PPCK1 will lead to the altered accumulation and periodicity of core circadian clock-related transcripts⁴⁰. In pineapple, AccPPCK2 was found to exhibit greater mRNA abundance than AccPPCK1, and AccPPCK2 also displayed diel mRNA abundance with high levels at night, suggesting that it functions in CAM¹⁵.

In the final metabolic step of phase I, the OAA formed as a result of PEP carboxylation is reduced to malate by NAD(P)-dependent malate dehydrogenase (MDH). Fourteen genes in pineapple encode MDH: three genes (AccMDH4, AccMDH5, and AccMDH8) are predicted to be cytosolic-localized and strongly expressed in leaves, suggesting their potential to perform functional roles in CAM; four genes (AccMDH10, AccMDH11, AccMDH12, and AccMDH13) are tandemly duplicated and lowly expressed except AccMDH13¹⁵.

In Arabidopsis, the malate is transported into the vacuole by an inward-rectifying anion-selective ion channel belonging to the aluminium-activated malate transporter (ALMT) family⁴¹. In K. fedtschenkoi, a putative ALMT6 gene (Kaladp0062s0038) displays diel mRNA abundance in leaves¹⁸. There are eight candidate ALMT family genes in pineapple, including three ALMT9 genes (AccALMT9-1–3) and five ALMT1 genes (AccALMT1-1–5). Only two ALMT9 genes (AccALMT9-1 and AccALMT9-3) showed high abundant transcript levels in photosynthetic leaf tissues. ALMT1 only has higher steady-state transcript levels at the midday on day 9 of water limitation in T. triangulare²³. The malate then undergoes protonation, with protons supplied by the tonoplast H⁺-ATPase and H⁺-PPiase, and is stored as malic acid. In the daytime, malic acid is effluxed out of the vacuole possibly through a putative tonoplast dicarboxylate transporter (tDT)⁴². There are five DT genes (AccDT1–5) in the pineapple genome, and AccDT2 and AccDT3 display specifically high abundant transcripts in daytime in photosynthetic leaf tissues, indicating that they may play a role in malic acid efflux in CAM. Decarboxylation of the malate during phase III of the CAM cycle occurs in pineapple primarily via PEP carboxykinase (PCK)^30,43, which, following oxidation of malate to OAA by NAD(P)-dependent MDH, decarboxylates OAA to PEP. A single PCK gene (AccPCK1) is present in the pineapple genome and it is predicted to encode a cytosolic enzyme¹⁵. It is an ortholog of AtPCK1 (AT4G37870.1), one of two PCK genes in Arabidopsis, which is expressed in guard cells and is implicated in stomatal closure⁴⁴. Despite the fact that extractable PCK activity from pineapple leaves is over 15 times higher than that of the malic enzymes (MEs)⁴⁵, and it remains possible that malate may also be decarboxylated, in part, by ME in pineapple⁴⁶. The comosus pineapple genome contains five ME genes encoding both NAD- and NADP-ME (Supplementary Table S3): two NADP-ME genes (AccNADP-ME1 and AccNADP-ME3) exhibit higher mRNA levels during the daytime in photosynthetic leaf tissues and one additional NADP-ME gene (AccNADP-ME2) shows none mRNA transcript in leaves; two NAD-ME genes (AccNAD-ME1 and AccNAD-ME2) encoding isoforms predicted to be localized to the mitochondria exhibit moderate abundant mRNA expression and AccNAD-ME2 also displayed higher mRNA level during the daytime¹⁵.

The abundant transcript level for ME genes in pineapple suggests that malate decarboxylation also results in the formation of pyruvate, which must then be phosphorylated to PEP by pyruvate phosphate dikinase (PPDK). Consistent with this supposition, a single candidate PPDK1 gene (AccPPDK1) was identified in the pineapple genome¹⁵, providing the metabolic flexibility to allow gluconeogenesis via both the PCK and ME/PPDK routes⁴⁷. AccPPDK1 displayed higher transcript abundance during the daytime. The AtPPDK1 gene encodes an enzyme predicted to be localized to the cytosol, but this enzyme might be localized to either the chloroplast or the cytosol depending upon the production of alternative transcripts arising from two different promoters⁴⁸. More detailed examination of this locus in pineapple is needed to verify this possibility. Overall, the enzymes making up the carboxylation and decarboxylation pathways in the CAM cycle in pineapple are encoded by gene families that are generally smaller than those encoded by the A. thaliana genome, because pineapple has one fewer whole-genome duplications than that have been reported for Arabidopsis and the grass family⁴⁹.

Circadian clock-associated cis-elements in CAM genes

In most living organisms, internally synchronized circadian clocks make it possible for them to coordinate behavior and physiology corresponding with the 24 h light-dark cycle. CCA1 and LATE ELONGATED HYPOCOTYL (LHY), two single-MYB domain transcription factors, are central to the circadian oscillator of angiosperms^50,51. CCA1 and LHY are morning expressed genes. They act to suppress the expression of the DNA sequence they bind to. CCA1 and LHY are partially redundant, and they can directly bind to the TIMING OF CAB EXPRESSION 1 (TOC1) also known as PRR1 (PSEUDO-RESPONSE REGULATOR 1) promoter to negatively regulate its expression⁵².

Circadian control of CAM has been implicated as a core component in diel re-programming of metabolism in CAM plants^20,53. A comprehensive spatial and temporal survey of gene co-expression clusters in pineapple leaf tissues reveals CAM pathway genes are enriched with clock-associated cis-elements, suggesting circadian regulation of CAM^15,16. At dawn, CCA1 and LHY repress evening-phased genes by binding to CBS and EE⁴⁹. In addition to CBS and EE, the G-box is also enriched in the CCA1 binding regions^54,55. TOC1 can bind to MOE as a negative regulator⁵⁶. In pineapple, all of the three βCA genes contain CBS in their promoter regions (Table 3), suggesting they may have function in βCA genes’ nighttime and early-morning transcripts abundance pattern in photosynthetic leaf tissues. All three copies of PPC genes also contain CBS in their promoter regions, along with MOE or G-box (Table 3). Interestingly, CAM pathway genes in A. comosus var. comosus, contain more circadian clock cis-regulatory elements than A. comosus var. bracteatus (Table 3). Besides the core CAM genes, more than 40% of transcription factors and transcription co-regulators displayed diel rhythmic expression in pineapple, suggesting it is a global adaptation⁵⁷. In a recent work by Heyduk and colleagues (2018), they demonstrated that some canonical CAM genes were unaltered by comparative transcriptomic analyses between the C₃ and CAM Erycina species. However, 149 gene families, including genes involved in light and ABA signaling, had significant differences in network connectivity, indicating that transcriptional cascades changes are critical for the transition from C₃ to CAM in Erycina²².

Table 3.

Cis-elements annotated at promoter regions of selected CAM photosynthetic genes in pineapple.

CAM enzyme	A. comosus var. comosus		A. comosus var. bracteatus
	Gene ID	TF binding motif	Gene ID	TF binding motif
βCA	Aco002732	CBS (2)	CB5.v30091940	CBS (2), G-Box (3)
	Aco005402	CBS (1)	CB5.v30297520	CBS (2)
	Aco006181	CBS (2)	CB5.v30069370	–
PPC	Aco010025	EE (1), MOE (2), CBS (2)	CB5.v30072230	–
	Aco018093	MOE (2), CBS (1)	CB5.v30160110	EE (1), MOE (1)
	Aco016429	G-box (1), CBS (1)	CB5.v30185780	–
			CB5.v30098990	G-box (1)
PPCK	Aco010095	G-box (1)	CB5.v30291670	–
	Aco013938	G-box (1)	CB5.v30308180	G-box (3)
MDH	Aco006122	CBS (1)	CB5.v30030450	–
	Aco007734	CBS (2)	CB5.v30063990	–
	Aco013935	MOE (2)	CB5.v30119070	–
	Aco002885	MOE (2), G-box (1)	CB5.v30217860	–
	Aco004349	MOE (2), G-box (1)	CB5.v30283140	CBS (2), G-box (2)
	Aco014690	G-box (2)	CB5.v30016950	CBS (1)
	Aco017525	CBS (3)	CB5.v30081100	MOE (1)
	Aco017526	–	CB5.v30308160	MOE (2)
	Aco017527	CBS (1)	CB5.v30167990	–
	Aco017528	CBS (2)	CB5.v30057750	EE (1), CBS (1)
	Aco019631	CBS (1)	CB5.v30057770	–
	Aco010232	MOE (1), G-box (1)	CB5.v30175150	CBS (1)
	Aco004996	MOE (3)	CB5.v30175160	CBS (2)
	Aco008626	–	CB5.v30057740	CBS (1)
			CB5.v30057760	G-box (1)
NAD_ME	Aco016569	–	CB5.v30106390	–
	Aco007622	CBS (1), G-box (1)	CB5.v30132860	CBS (1), G-box (2)
NADP_ME	Aco009967	G-box (4), MOE (1)	CB5.v30038850	G-box (2)
	Aco005631	CBS (1)	CB5.v30285200	CBS (1)
	Aco005989	G-box (2)	CB5.v30300470	G-box (3), ME (1)
PCK	Aco017762	MOE (2), G-box (1), CBS (1)	CB5.v30124740	MOE (1), G-box (1), CBS (1)
PPDK	Aco024818	EE (2), G-box (1), CBS (1)	CB5.v30137360	EE (1), G-box (1), CBS (1)

Evolution of CAM photosynthesis

C₄ and CAM photosynthesis are innovations that evolved in response to decreasing atmospheric levels of CO₂ and water-limiting environments^2,9. CAM has a higher incidence³, and mutation of CAM genes in CAM species is not lethal^40,58. Both C₄ and CAM have evolved independently multiple times, even within individual families, or even genera during angiosperm evolution^59–61. For example, in the Neotropical family Bromeliaceae, to which pineapple belongs, CAM photosynthesis evolved independently at least four, and probably five times⁵⁹.

Recruitment of pre-existing mechanisms underlying C₃ photosynthesis is adopted in Gynandropsis gynandra (referred to previously as Cleome gynandra), a C₄ plant which is relatively closely related to Arabidopsis⁶². Furthermore, gene duplication also plays a profound role in the evolution of C₄. For example, βCA genes are tandemly duplicated in sorghum⁶³. After duplication, some C₄ genes, such as C₄ PPC genes, NADP-MDH genes, and PPDK genes, underwent adaptive evolution⁶³.

Comparative analyses demonstrated signatures of convergence in protein sequence and re-scheduling of diel transcript abundance of genes involved in nocturnal CO₂ fixation, stomatal movement, heat tolerance, the circadian clock, and carbohydrate metabolism^14,18,21. Firstly, convergent evolution has been detected in terms of diel cycles of gene transcript abundance¹⁸. PPCK is a key regulator of PPC, which can activate PPC by phosphorylating it. Both AccPPCK2 and KfPPCK2 showed diel expression patterns¹⁸. Secondly, a convergent amino-acid change in PPC2 was discovered to be shared by K. fedtschenkoi and P. equestris and the PPC2 gene in K. fedtschenkoi is a much lower abundance transcript relative to the CAM-associated PPC1 gene, so the function of PPC2 has yet to be linked to CAM directly in either K. fedtschenkoi or P. equestris¹⁸.

These findings are consistent with the hypothesis that the CAM photosynthesis evolved as a result of a re-organization of pre-existing metabolic pathways^11,15. These different features were later coordinated to form the functional CAM photosynthesis.

Concluding remarks

Genomic studies have led to a renaissance in CAM research. Recent genomic and transcriptomic information from CAM species has improved our understanding of the evolution of CAM photosynthesis^14–21. The identified candidate genes provide initial targets for detailed functional studies of how the CAM genes have evolved through regulation of gene expression to gain the observed spatial and temporal expression patterns, and loss of repressors is certainly involved. It may be possible for us to apply genome editing to verify functions of candidate CAM genes. CRISPR/Cas9 technology will be a powerful tool to get higher order mutants of tandemly duplicated genes in the same chromosome, which is impossible to generate by traditional mutagenesis methods.

Water loss from stomata for C₃ plants can be very substantial under hot and dry condition. Adjusting the temporal pattern of stomatal movement genes may be a key evolutionary step for switching stomatal opening from the light period to dark³². Enrichment of different sets of circadian clock regulatory cis elements may have played a role in this dramatic shift in gene regulation in pineapple and P. equestris. CAM photosynthesis and its associated high WUE are key evolutionary innovations that adapted to arid environments and/or low CO₂ environment and this valuable trait is a direct consequence of stomatal closure throughout hottest and driest part for the 24 h cycle, and leaf succulence.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary information

Supplementary Information accompanies this paper at (10.1038/s41438-019-0229-0).